Process for the preparation of (S) - or (R) -3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid

ABSTRACT

The present invention relates to a novel process for the preparation of (S)- or (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid and to novel microorganisms capable of utilizing the propionamide of the formula 
                         
in the form of the racemate or of its optically active isomers as the sole nitrogen source.

The present application is a divisional of application Ser. No.09/214,679, filed Dec. 30, 1999 (now U.S. Pat. No. 6,773,910), which isa 371 U.S. national phase of PCT/EP97/03670, filed Jul. 10, 1997.

The present invention relates to a novel process for the preparation of(S)- or (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid and tonovel microorganisms capable of utilizing the propionamide of theformula

in the form of the racemate or of its optically active isomers as thesole nitrogen source.

(S)-3,3,3-Trifluoro-2-hydroxy-2-methylpropionic acid is an importantintermediate for the preparation of therapeutic amides (EP-A 0 524 781).

In the following text, 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acidis abbreviated to 2,2-HTFMPS, and3,3,3-trifluoro-2-hydroxy-2-methyl-propionamide to 2,2-HTFMPA.

In J. Chem. Soc., 1951, p. 2329 there is described a process for thepreparation of (S)-2,2-HTFMPS where the corresponding racemate isconverted into the desired (S) enantiomer by means ofdimethoxystrychnine. The disadvantage of this process is thatdimethoxystrychnine, which is employed for the racemate resolution, istoo expensive.

EP-A 0 524 781 describes a process for the preparation of (S)-HTFMPS, inwhich the corresponding racemate is converted into the desired (S)enantiomer by means of (S)-(−)-α-methylbenzylamine. The disadvantage ofthis process is that large amounts of (S)-(−)-α-methylbenzylamine mustbe employed, which, again, makes this process too expensive.

It is an object of the present invention to provide an inexpensive,technically feasible process for the preparation of (S)- or(R)-2,2-HTFMPS.

This object is achieved by the microorganisms according to claim 1 andclaim 11 according to the invention, the polypeptides according to claim4 and by the processes according to claims 15 and 16.

Accordingly, the present invention relates to microorganisms selectedfrom the wild, so-called “wild types”, enzyme extracts therefrom,enzymes isolated therefrom having stereospecific amidohydrolaseactivity, and DNA/DNA fragments which are isolated from the “wild types”and which encode a stereospecific amidohydrolase. The present inventionfurthermore relates to so-called genetically engineered microorganismscomprising these DNA fragments, or vectors. A further subject-matter isa process for the preparation of (S)- or (R)-2,2-HTFMPS and a processfor the preparation of (S)- or (R)-2,2-HTFMPA using the above-describedmicroorganisms.

The invention is illustrated in greater detail by the Figures below.

FIG. 1 shows the restriction map of the isolated DNA

FIG. 2 shows plasmid pPRS1b

FIG. 3 shows plasmid pPRS2a

FIG. 4 shows the pH optimum of the amidohydrolase

FIG. 5 shows the Michaelis-Menten kinetics of the amidohydrolase

FIG. 6 shows the temperature optimum of the amidohydrolase

FIG. 7 shows the effect of methanol on the amidohydrolase

The “wild types” according to the invention can be isolated from soilsamples, sludge or waste water with the aid of customary microbiologicaltechniques. In accordance with the invention, the isolation is performedin such a way that these are cultured in the customary manner in amedium comprising the propionamide of the formula VI in the form of theracemate or one of its optically active isomers as the sole nitrogensource, together with a suitable carbon source. Then, those which arestable and which utilize the propionamide of the formula VI as the solenitrogen source are selected from the culture obtained by culturing.

By way of suitable carbon sources, the “wild types” are capable ofutilizing sugar, sugar alcohols or carboxylic acids as growth substrate.Examples of sugars which can be used are glucose, arabinose, rhamnose,lactose or maltose. Sugar alcohols which can be used are, for example,sorbitol, mannitol or glycerol. Citric acid is an example of acarboxylic acid which can be used. Glycerol or glucose is preferablyemployed as the carbon source.

The selection and growth media which can be used are thoseconventionally used in expert circles, such as, for example, a mineralsalt medium as described by Kulla et al., Arch. Microbiol. 135, pp. 1–7,1983.

It is expedient to induce the active enzymes of the microorganismsduring growth and selection. The propionamide of the formula VI in theform of the racemate or one of its optically active isomers, acetamideor malonic diamide, can be used as the enzyme inductor.

Growth and selection normally take place at a temperature from 0 to 42°C., preferably from 20 to 37° C. and at a pH of 4 to 9, preferably at apH of 6 to 8.

Preferred “wild types” are those of the genus Klebsiella, Rhodococcus,Arthrobacter, Bacillus and Pseudomonas which utilize propionamide(formula VI). Very especially preferred are microorganisms of thespecies Klebsiella oxytoca PRS1 (DSM 11009), Klebsiella oxytoca PRS1K17(DSM 11623), Pseudomonas sp. (DSM 11010), Rhodococcus opacus ID-622 (DSM11344), Arthrobacter ramosus ID-620 (DSM 11350), Bacillus sp. ID-621(DSM 11351), Klebsiella planticula ID-624 (DSM 11354) and Klebsiellapneumoniae ID-625 (DSM 11355), and their functionally equivalentvariants and mutants. The Klebsiella oxytoca (DSM 11009), Klebsiellaplanticula ID-624 (DSM 11354) and Klebsiella pneumoniae ID-625 (DSM11355) “wild types” preferentially have (R)-amidohydrolase activity, andthe Pseudomonas sp. (DSM 11010), Rhodococcus opacus ID-622 (DSM 11344),Arthrobacter ramosus ID-620 (DSM 11350) and Bacillus sp. ID-621 (DSM11351) “wild types” preferentially have (S)-amidohydrolase activity. Themicroorganisms termed DSM 11010, DSM 11009 were deposited on 24.06.1996,the microorganisms termed DSM 11355, DSM 11354 on 27.12.1996, themicroorganisms termed DSM 11351, DSM 11350 and DSM 11344 on 13.12.1996and the microorganisms termed DSM 11623 on 20.06.1997 at the DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroderweg 1b,D-38124 Braunschweig in compliance with the Budapest Treaty.

“Functionally equivalent variants and mutants” of the “wild types” areto be understood as meaning strains which have essentially the samecharacteristics and functions as the original microorganisms. Suchvariants and mutants may be formed randomly, for example by UVirradiation, or in a directed fashion by chemical mutagenesis, forexample by intercalating substances, such as acridine dyes.

Taxonomic description of Klebsiella oxytoca PRS1 (DSM 11009) Cell shapeRods Width μm 1.0–1.2 Length μm 1.2–2.0 Motility − Gram reaction − Lysisby 3% KOH + Aminopeptidase (Cerny) + Spores − Oxidase − Catalase +Growth + anaerobic Gas from glucose + Acid from (ASA) Glucose +Fructose + Xylose + Erythritol − Adonitol + D-Mannose + L-Rhamnose +Inositol + Sorbitol + α-Methyl-D-glucoside + Cellobiose + Maltose +Lactose + D-Arabitol + ONPG + ADH − LDC w ODC − VP + Indole + H₂Sgeneration − Simmons citrate + Urease + Methyl Red − Hydrolysis ofGelatin − DNA − Tween 80 −

Taxonomic description of Pseudomonas sp. (DSH 11010) Cell shape RodsWidth μm 0.7–0.8 Length μm 1.5–3.5 Motility + Gram reaction − Lysis by3% KOH + Aminopeptidase (Cerny) + Spores − Oxidase + Fluorescence +Catalase + Growth at 41° C. − ADH + Urease − Hydrolysis of gelatin +Nitrate reduction − Denitrification − Levan from sucrose + Lecithinase +Substrate utilization Adipate − Citrate + Malate + L-Mandelate − Phenylacetate − D-Glucose + Maltose − Trehalose + Mannitol + Adonitol +Acetamide + Hippurate − Tryptamine − Butylamine − Abbreviations: ASA:acetylsalicylic acid ONPG: O-Nitro-phenylgalactosidase ADH: Alcoholdehydrogenase LDC: Lactate decarboxylase ODC: Ornithin decarboxylase VP:Voges Proskauer

The enzyme according to the invention which has stereospecificamidohydrolase activity can be obtained, for example, from the “wildtypes” which have already been described and are capable of hydrolysingthe propionamide of the formula

in the form of the racemate or its (R) isomers, and functionallyequivalent variants and mutants thereof.

“Functionally equivalent variants and mutants” of the enzymes are to beunderstood as meaning enzymes which essentially have the samecharacteristics and functions. Such variants and mutants can be formedrandomly, for example by mutation.

The enzyme is expediently characterized by

-   a) a pH optimum of pH 10±0.5-   b) a temperature optimum of between 65 and 70° C. at a pH of 10 and-   c) a K_(M) value for the substrate (R)-2,2-HTFMPA of 32 mM (60° C.    in 100 mM CAPS buffer (3-(cyclohexylamino)-1-propanesulphonic acid)    pH 10), in particular in that-   d) a methanol concentration of 5 to 20% has an inhibitory effect and-   e) the N-terminal amino acid sequence is:    Met-Lys-Trp-Leu-Glu-Glu-Ser-Ile-Met-Ala-Lys-Arg-Gly-Val-Gly-Ala-Ser-Arg-Lys-Pro.

This stereospecific amidohydrolase can be isolated from theabove-described “wild types” which are capable of utilizing thepropionamide of the formula VI in the form of the racemate or of its Risomer as the sole nitrogen source. The amidohydrolase is expedientlyisolated from the “wild types” of the genus Klebsiella, preferably fromKlebsiella oxytoca PRS1 (DSM 11009) or Klebsiella oxytoca PRS1K17 (DSM11623).

Naturally, this enzyme may also be isolated from the geneticallyengineered microorganisms which are derived from these “wild types”.

To obtain the stereospecific amidohydrolase, the “wild types” are grown(cultured) in the customary manner in an aqueous nutrient mediumcomprising a carbon source, a nitrogen source, mineral salts and avitamin source. The “wild types” are expediently cultured at atemperature from 20 to 35° C. and a pH of 6 to 8. The enzyme can then beisolated by enzyme purification methods known per se after celldisruption, for example using the French press.

The DNA according to the invention, or the DNA fragments according tothe invention, which encode a stereospecific amidohydrolase as it isshown, in particular, by the amino acid sequence in SEQ ID No. 2 andwhich are characterized by the restriction map as shown in FIG. 1 and,in particular, by the nucleotide sequence in SEQ ID No. 1, also embracetheir functionally equivalent genetic variants and mutants, i.e. geneswhich are derived from the genes of the wild-type organisms and whosegene products are essentially unmodified with regard to their biologicalfunction. The functionally equivalent genetic variants and mutants thusembrace, for example, base exchanges within the scope of the knowndegeneration of the genetic code, as they can be generated, for example,artificially to adapt the gene sequence to the preferred codon usage ofa particular microorganism in which expression is to take place. Thegenetic variants and mutants also embrace deletions, insertions andsubstitutions of bases or codons, as long as the gene products of genesmodified in this way remain essentially unaltered with regard to theirbiological function. This embraces, for example, gene sequences whichexhibit a high level of homology to the wild-type sequences, for examplegreater than 70%, and which are capable of hybridizing with thecomplement of the wild-type sequences under stringent hybridizationconditions, for example at temperatures between 60 and 70° C. and at asalt content of 0.5 to 1.5 M, in particular at a temperature of 67° C.and a salt content of 0.8 M.

The above-described “wild types” which are employed as starting materialfor isolating the stereospecific amidohydrolase according to theinvention may be employed as starting material for the DNA according tothe invention.

The intact genes, or the intact DNA fragments according to theinvention, can be isolated by known methods starting from a gene libraryfor suitable microorganisms, such as Klebsiella oxytoca, from which theamidohydrolase gene, or fragments thereof, can be isolated and cloned ina known manner by hybridization with labelled oligonucleotides whichcontain sub-sequences of the amidohydrolase genes. The amidohydrolasegene will be abbreviated to sad hereinbelow.

To improve transcription, the sad gene is advantageously placed underthe control of a strong promoter. The choice of promoter depends on thedesired expression conditions, for example on whether constitutive orinduced expression is desired, or on the microorganism in whichexpression is to take place.

Suitable promoters are the promoters P_(L) and P_(R) of phage lambda(cf. Schauder et al., Gene, 52, 279–283, 1987), the P_(trc) promoter(Amann et al., Gene, 69, 301–315, 1988), the promoters P_(Nm), P_(Sl),(M. Labes et al., Gene, 89, 37–46, 1990), the P_(trp) promoter (Amann etal., Gene, 25, 167–178, 1983), the P_(lac) promoter (Amann et al., Gene,25, 167–178, 1983) and the P_(tac) promoter, a hybrid of theabovementioned P_(trp) and P_(lac) promoters, which can be employed asconstitutive or inducible promoters (Russel and Bennett, Gene, 20,231–243, 1982). The P_(lac) promoter is preferably used.

For use in the production of, for example, (R)-2,2-HTFMPS in a suitableproduction strain, the DNA fragments according to the invention areexpediently incorporated into suitable known vectors, preferablyexpression vectors, with the aid of known techniques. Autonomously andself-replicating plasmids or integration vectors may be used as vectors.

Depending on the type of vector chosen, the sad genes can be expressedin a variety of microorganisms. Suitable vectors are both vectors with aspecific host range and vectors with a broad host range. Examples ofvectors with a specific host range, for example for E. coli, are pBR322(Bolivar et al., Gene, 2, 95–113), the commercially availablepBLUESCRIPT-KS+®, pBLUESCRIPT-SK+® (Stratagene), pUC18/19(Yanisch-Perron et al., Gene 33, 103–119, 1985), pK18/19 (Pridmore,Gene, 56, 309–312, 1987), pRK290X (Alvarez-Morales et al., Nucleic AcidsResearch, 14, 4207–4227) and pRA95 (available from Nycomed Pharma AS,Huidove, Denmark). pBLUESCRIPT-KS+® is preferably employed.

All vectors which are suitable for Gram-negative bacteria may beemployed as broad host-range vectors.

Examples of such broad host-range vectors are pRK290 (Ditta et al.,PNAS, 77, 7347–7351, 1980) or their derivatives, pKT240 (Bagdasarian etal., Gene, 26, 273–282, 1983) or its derivatives, pGSS33 (Sharpe, Gene,29, 93–102, 1984), pVK100 (Knauf and Nester, Plasmid, 8, 45–54, 1982)and its derivatives, pME285 (Haas and Itoh, Gene, 36, 27–36, 1985) andits derivatives.

For example the plasmids pPRS1b (FIG. 2), pPRS2a (FIG. 3), pPRS4 andpPRS7 were obtained in this manner.

To generate the production strains for fermentation, i.e. strains whichcan be employed for the preparation of, for example, (R)-2,2-HTFMPS, thevectors or DNA fragments according to the invention must be introducedinto the desired host strains which are suitable for expression. To thisend, the microorganisms are expediently transformed with the vectorscontaining the DNA fragments according to the invention in the customarymanner which is known per se. Then, the microorganisms can contain theDNA fragment according to the invention either on a vector molecule orintegrated in their chromosome.

Suitable host strains, preferably strains with a high substrate andstarting material tolerance are, for example, microorganisms of thegenus Pseudomonas, Comamonas, Bacillus, Rhodococcus, Acinetobacter,Rhizobium, Agrobacterium, Rhizobium/Agrobacterium or Escherichia, thelatter ones being preferred. Especially preferred are the microorganismsEscherichia coli DH5, Escherichia coli XLl-Blue® and Escherichia coliXL1-Blue MRF′®. Examples of suitable production strains are thusmicroorganisms of the species Escherichia coli DH5 and Escherichia coliXL1-Blue MRF′®, each of which contains plasmid pPRS1b, pPRS2a, pPRS4 orpPRS7.

The microorganism Escherichia coli XL1-Blue MRF′®/pPRS2a was depositedas DSM 11635 on 30.06.1997 at the Deutsche Sammlung fur Mikroorganismenund Zellkulturen GmbH, D-38124 Braunschweig, Mascheroderweg 1b incompliance with the Budapest Treaty.

The transformed host strains (production strains) can be isolated from aselective nutrient medium supplemented with an antibiotic to which thestrains are resistant due to a marker gene located on the vector or theDNA fragment.

The process according to the invention for the preparation of (S)- or(R)-2,2-HTFMPS of the formulae

and/or of (R)- or (S)-2,2-HTFMPA of the formulae

comprises the conversion of the propionamide of the formula

by means of the above-described microorganisms according to theinvention, or by means of the enzymes isolated therefrom which exhibitstereospecific amidohydrolase activity.

The process for the preparation of (R)-2,2-HTFMPS and/or of(S)-2,2-HTFMPA is expediently carried out using the “wild types” of thegenus Klebsiella, preferably of the species Klebsiella oxytoca PRS1 (DSM11009), Klebsiella oxytoca PRS1K17 (DSM 11623), Klebsiella planticulaID-624 (DSM 11354), Klebsiella pneumoniae ID-625 (DSM 11355), using thegenetically engineered microorganisms derived from these “wild types” orusing the enzyme having a stereospecific amidohydrolase activity.

The process for the preparation of (S)-2,2-HTFMPS and/or (R)-2,2-HTFMPAis expediently carried out using the “wild types” of the genusPseudomonas, Rhodococcus, Arthrobacter or Bacillus, in particular thespecies Pseudomonas sp. (DSM 11010), Rhodococcus opacus ID-622 (DSM11344), Arthrobacter ramosus ID-620 (DSM 11350) and Bacillus sp. ID-621(DSM 11351).

The biotransformation can be performed on dormant cells (non-growingcells which no longer require a carbon and energy source) or on growingcells, after having grown the microorganisms in the customary manner.The biotransformation is preferably carried out on dormant cells.

Media conventionally used by those skilled in the art may be employedfor the biotransformation, such as, for example, phosphate buffers oflow molarity, HEPES buffers, or the above-described mineral salt medium.

The biotransformation is expediently carried out with the single orcontinuous addition of propionamide (formula VI) in such a way that theconcentration does not exceed 10% by weight, preferably 2.5% by weight.

The pH of the medium can range from 4 to 10, preferably from 5 to 9.5.The biotransformation is expediently carried out at a temperature of 10to 60° C., preferably 20 to 40° C.

The resulting (S)- or (R)-2,2-HTFMPS, or (S)- or (R)-2,2-HTFMPA,respectively, can be isolated by customary work-up methods, such as, forexample, by extraction.

The yield of (S)- or (R)-2,2-HTFMPS, or (S)- or (R)-2,2-HTFMPA,respectively, can be improved further in the customary manner by varyingthe nutrients in the medium and by adapting the fermentation conditionsto the microorganism in question.

If appropriate, the (S)- or (R)-2,2-HTFMPA is hydrolysed to give thecorresponding acid, either chemically in the presence of a base ormicrobiologically using microorganisms of the genus Rhodococcus.

An alkali metal hydroxide may be employed as the base. Sodium hydroxideor potassium hydroxide is expediently employed as the alkali metalhydroxide.

The microbiological hydrolysis is expediently carried out usingmicroorganisms of the species Rhodococcus equi, Rhodococcus rhodochrousor Rhodococcus sp. S-6, preferably using microorganisms of the speciesRhodococcus equi TG 328 (DSM 6710) or its functional equivalent variantsand mutants. The microorganism Rhodococcus equi TG 328 is described inU.S. Pat. No. 5,258,305 and was deposited on Sep. 13, 1991 at theDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, D-38124Braunschweig, Mascheroderweg 1b in compliance with the Budapest Treaty.Normally, these microorganisms are grown by the method of Gilligan etal. (Appl. Microbiol. Biotech., 39, 1993, 720–725) before the actualmicrobiological hydrolysis is carried out. In principle, themicrobiological hydrolysis is effected by methods conventionally used inthe art. The hydrolysis is expediently effected at a temperature of 20to 40° C. and a pH of 6 to 9.

The propionamide of the formula

is prepared in such a manner that, in a first step, trifluoroacetate ofthe formula

is first converted into trifluoroacetone of the formula

using a mineral acid.

Examples of a mineral acid which can be employed are hydrochloric acid,sulphuric acid, nitric acid or phosphoric acid. Acids which arepreferably employed are sulphuric acid, phosphoric acid or nitric acid,in particular sulphuric acid.

The first step of the reaction is expediently carried out in a polarprotic solvent such as, for example, in a lower alcohol, in water or ina mixture of lower alcohol/water. Lower alcohols which can be employedare, for example, methanol, ethanol, propanol, isopropanol, butanol,tert-butanol or isobutanol.

The first step of the reaction is expediently carried out at atemperature of 50 to 100° C., preferably at a temperature of 70 to 95°C.

In the second step of the process according to the invention,trifluoroacetone (formula IV) is reacted with a cyanide t o give thepropionitrile of the formula

Cyanides which are expediently employed are alkali metal cyanides suchas sodium cyanide or potassium cyanide, preferably sodium cyanide.

The second step of the reaction is expediently carried out in thepresence of a mineral acid. Suitable mineral acids are those which havebeen described above. The preferred mineral acid is sulphuric acid.Normally, an excess of mineral acid is employed, based ontrifluoroacetone. It is preferred to use 1 to 10 mol of mineral acid permole of trifluoroacetone. The solvents which can be used are the same asin the first step.

The second step is expediently carried out at a temperature of −20 to100° C., preferably 0 to 20° C.

In the third step of the process according to the invention, thepropionitrile of the formula V is converted into the propionamide of theformula VI, either chemically in a concentrated mineral acid ormicrobiologically using mutated microorganisms of the genus Rhodococcus.

Mineral acids which can be employed are the same as in the first andsecond step. A “concentrated mineral acid” is to be understood asmeaning hereinbelow a 30 to 100% strength mineral acid. A 75 to 100%strength, preferably a 90 to 100% strength, mineral acid is expedientlyused in the third step. The chemical reaction in the third step isexpediently carried out at a temperature of 0 to 160° C., preferably 70to 120° C.

The mutated microorganisms of the genus Rhodococcus no longer containamidase and are thus no longer capable of converting an amide into thecorresponding acid. The mutation can be effected by customary methods(J. H. Miller, Experiments in Molecular Genetics, Cold Spring HarborLaboratory, 1972, p. 24). Expedient mutation methods are the frameshiftmethod, the deletion method or the transposon insertion method.

Suitable microorganism species for the mutation are Rhodococcus equi,Rhodococcus rhodochrous or Rhodococcus sp. S-6. It is preferred tomutate the above-described Rhodococcus equi TG 328 (DSM 6710), thusobtaining Rhodococcus equi TG 328-2 (DSM 11636) and its functionallyequivalent variants and mutants. The microorganism TG 328-2 wasdeposited on 30.06.1997 at the Deutsche Sammlung für Mikroorganismen undZellkulturen GmbH, D-38124 Braunschweig, Mascheroderweg 1b in compliancewith the Budapest Treaty. This microorganism is cultured under the sameconditions as the unmutated microorganisms which have already beendescribed above.

(R)- and (S)-2,2-HTFMPA are compounds hitherto not described in theliterature and therefore also part of the invention. They can beemployed as novel intermediates for the preparation of (R)- or(S)-2,2-HTFMPS, for example by hydrolysis in the presence of a base.

EXAMPLE 1

Preparation of Trifluoroacetone

500 g (4.9 mol) of concentrated sulphuric acid (96% strength; Merck)were added to 1 l of distilled water, and the mixture was heated to 73°C. Then, 500 g (2.69 mol) of trifluoroacetate were added slowly, duringwhich process two phases formed. The batch was heated to refluxtemperature, and the trifluoroacetone formed in the process wasdistilled off. After 2 hours, 293.8 g of trifluoroacetone were isolatedas colourless liquid, corresponding to a yield of approx. 90%. GCanalysis revealed a purity of 92.1%.

EXAMPLE 2

Preparation of 2-hydroxy-2-methyl-3,3,3-trifluoro-methylpropionitrile

39.4 g of sodium cyanide (0.763 mol) were added to 174 ml of distilledwater and the mixture was cooled to −1° C. 100 g of trifluoroacetone(0.822 mol) were subsequently added dropwise, during which process thetemperature of the reaction mixture climbed to 6° C. After addition oftrifluoroacetone had ended, 293.4 g of 6 N sulphuric acid (1.4916 mol ofH) were added at 4–5° C. The reaction mixture was then stirred overnightat room temperature. The batch was subsequently extracted with ethylacetate or with tert-butyl methyl ether and the combined organic phaseswere distilled either under atmospheric pressure at 32° C. or underslightly subatmospheric pressure (300–120 mbar). In total, 88 g ofproduct of 91.2% purity (measured by GC) were obtained, whichcorresponds to a yield of 75.6%.

EXAMPLE 3

a) Chemical Preparation of (R,S)-2,2-HTFMPA

98% strength sulphuric acid was introduced into the reaction vesselunder argon atmosphere. 15 g of2-hydroxy-2-methyl-3,3,3-trifluoromethylpropionitrile (86.9% accordingto GC) were added to this, and the reaction mixture was heated to 95° C.After the addition of starting material, the reaction mixture was heatedfor 15 minutes at 114° C. The reaction mixture was then cooled to 5° C.,during which process a viscous brown solution formed. 40 g of distilledwater were subsequently added dropwise. During this process, care wastaken that the temperature of the reaction mixture did not exceed 15° C.The yellowish suspension formed in this process was cooled for 15minutes at −15° C. and then filtered. The filter cake was washed with 20ml of ice-cold water and then dried in vacuo. This gave 12.64 g of apale yellowish crude product. The crude product was subsequentlyrefluxed in 13 ml of ethyl acetate and then cooled to room temperature.This suspension was treated with 15 ml of hexane, and the mixture wascooled to 0° C. The mixture was then washed once more with hexane.Drying in vacuo gave 11.8 g of product, which corresponds to a yield of80.2%.

M.p.: 143.1–144.3° C.

b) Microbiological Production of (R,S)-2,2-HTFMPA (Using a MutatedMicroorganism of the Genus Rhodococcus)

For mutation purposes, Rhodococcus equi TG 328 was incubated by standardmethods overnight in “nutrient broth” at 30° C. with added acridine ICR191. The cells were then harvested and washed using 0.9% strength NaClsolution. The cells were then incubated in fresh medium overnight at 30°C.

The mutated cells were selected in a mineral salt medium described byGilligan et al. (Appl. Microbiol. Biotech., 39, 1993, 720–725) in thepresence of fluoroacetamide as counterselective agent. Thiscounterselective agent only destroys growing bacteria. Mutants, which nolonger contain amidase and no longer grow on (R,S)-2,2-HTFMPA surviveand are concentrated. The cells were subsequently harvested, washed with0.9% strength NaCl solution, incubated overnight in fresh medium andthen plated out. The colonies were tested for nitrile hydrataseactivity. The frequency of the desired mutation was 2%.

The mutant of Rhodococcus equi TG 328–2 was grown in a mineral saltmedium as described by Gilligan et al., (ibid). The washed cells wereincubated at OD_(650 nm)=5.0, both with2-hydroxy-2-methyl-3,3,3-trifluoromethylpropionitrile solution (1%strength) and with a (R,S)-2,2-HTFMPA solution (1% strength) in 100 mMphosphate buffer (pH 7.7) at 37° C. After 16 hours, GC analysisdemonstrated that the nitrile was converted quantitatively into theamide, whereas the amide was not hydrolysed to give the acid.

EXAMPLE 4

Production of (S)-2,2-HTFMPA and (R)-2,2-HTFMPS by Means of aMicroorganism Containing an Amidohydrolase (Wild Type)

4.1. Selection and Isolation of Microorganisms with (R)- and (S)-amidaseActivity

100 ml of phosphate buffer (0.1 M, pH 7.0) were added to a soil sampleof 10 g, and the mixture was left to stand for 10 minutes and filtered.Then, the supernatant (5.0 ml) or 1 ml of waste water (ARA, Visp) wassubcultured in a mineral salt medium (25 ml; Kulla et al., Arch.Microbiol. 135, pp. 1–7, 1983) containing glycerol and (R,S)-HTFMPA(carbon/nitrogen ratio 5:1). This culture was subsequently incubateduntil a mixed culture had formed which can utilize (R)- and/or(S)-2,2-HTFMPA as the sole nitrogen source. This culture was thensubcultured repeatedly and incubated at 30° C. until a mixed culture hadformed.

The pure culture of these microorganisms was maintained with the aid oftraditional microbiological techniques.

The resulting microorganism strains were then tested on agar plates forgrowth on (R,S)-2,2-HTFMPA. The positive strains were tested further.These strains were then used to inoculate a preculture medium. Themicroorganisms contained in this preculture were transferred into themineral salt medium and then tested for their capability of selectivelyutilizing (R)-2,2-HTFMPA and/or (S)-2,2-HTFMPA as sole nitrogen source,the supernatant being checked by GC for (R)-2,2-HTFMPS or (S)-2,2-HTFMPSformation and for the concentration of one of the two amide enantiomers.

4.2. Determination of (R)- or (S)-2,2-HTFMPA Amidohydrolase Activity

To determine the hydrolase activity, the microorganism suspension wasbrought to an optical density of 4.0 at 650 nm. A phosphate buffer (100mmolar), pH 7.0, supplemented with 0.5% by weight of (R,S)-HTFMPA, actedas the medium. This suspension was incubated for 2 hours at 30° C. withshaking. The NH₄ ⁺ liberated by the hydrolase was determined eithercalorimetrically or by means of an ammonium electrode, and the HTFMPAwas measured by GC. The activity was expressed as g of (R)- or(S)-HTFMPA converted/l/h/optical density at 650 nm, with the provisothat 1 mmol of NH₄ ⁺ formed equals 1 mmol of converted HTFMPA.

TABLE 1 Hydrolase activity of Klebsiella and Pseudomonas Hydrolaseactivity (g/l/h/O.D. 650 nm) Strain (R)-specific (S)-specific DSM 110090.11 — (Klebsiella oxytoca PRS1) DSM 11010 — 0.09 (Pseudomonas sp.)4.3. Production of (S)-2,2-HTFMPA and (R)-2,2-HTFMPS.

Klebsiella oxytoca PRS1 (DSM 11009), Klebsiella planticula ID-624 (DSM11354) or Klebsiella pneumoniae ID-625 (DSM 11355) were incubated for 2days at 30° C. on mineral salt medium agar plates with glycerol ascarbon source and (R,S)-2,2-HTFMPA as sole nitrogen source. Thecomposition of the mineral salt medium is described in Kulla et al.,Arch. Microbiol., 135, pp. 1–7, 1983. These plated microorganisms wereused to incubate a preculture medium of the same composition which wasincubated for 2 days at 30° C. The same mineral salt medium (600 ml) wasinoculated with 50 ml of preculture for induction and biomass productionand incubated at 30° C. for 21 hours. The cells were subsequentlyharvested by centrifugation and taken up in 0.1 M phosphate buffer pH7.0. After resuspending the cells in 0.05 M phosphate buffer (500 ml, pH8.0), an optical density at 650 nm of 10 was established, and 1.0% byweight of (R,S)-2,2-HTFMPA was added. After incubation for approx. 5.5hours at 40° C., (R)-2,2-HTFMPA was converted completely into thecorresponding acid, which corresponds to an optical purity (ee) of 100%and a yield of 48%.

The course of the reaction was monitored on the basis of NH₄ ⁺liberation and GC analysis of the supernatant.

4.4. Production of (S)-2,2-HTFMPS and (R)-2,2-HTFMPA Using aMicroorganism Containing an (S)-amidohydrolase

The microorganisms Pseudomonas sp. (DSM 11010), Rhodococcus opacusID-622 (DSM 11344), Arthrobacter ramosus ID-620 (DSM 11350) and Bacillussp. ID-621 (DSM 11351) were isolated analogously to Example 4.1. Theinduction period was 2 days, and all the other conditions were the sameas in Example 4.3.

In contrast to Example 4.3., the bio-transformation using thesemicroorganisms was carried out with 0.5% by weight of (R,S)-2,2-HTFMPA.The strain Pseudomonas sp. (DSM 11010) has an (S)-specific hydrolase,and the activity of the hydrolase at pH 6.0 was determined as 0.09 g of(S)-2,2-HTFMPA (ee=86%), converted/l/h/O.D. 650 nm.

4.5. Work-up of (S)-2,2-HTFMPA and (R)-2,2-HTFMPS

a) by Means of Extraction

196 ml of a reaction mixture containing (S)-2,2-HTFMPA and(R)-2,2-HTFMPS (obtained from Example 4.3), 0.1 M phosphate buffer (250ml), pH 10 were extracted 3 times with ethyl acetate (200 ml). Thecombined organic phases were dried with Na₂SO₄ and then evaporated at40° C. and 50 mbar. This gave 912 mg of moist product. This product wasdissolved in hot ethyl acetate (1.3 ml) and the solution was then cooledto room temperature. Addition of hexane (2 ml) resulted in precipitationof the product. The mixture was cooled to 0° C., and the product wasfiltered off and then dried in vacuo at 50° C. This gave 791 mg of(S)-2,2-HTFMPA, which corresponds to a yield of 78.2% based on half ofthe quantity employed. Only the (S) isomer was identified by means ofchiral GC analysis. The remaining aqueous phase was brought to pH 1 withconcentrated HCl and then extracted twice with ethyl acetate (200 ml)The extracts were evaporated at 40° C. and then dried. 1 ml of toluenewas then added, and the mixture was cooled to room temperature. Afurther 2 ml of hexane were added, and the mixture was cooled to 0° C.The solid was washed 2–3 times with hexane and then dried. In total, 664mg of (R)-2,2-HTFMPS were obtained from the aqueous phase after dryingin vacuo at 35° C., which corresponds to a yield of 65.7% based on halfof the amount employed. Only the (R) isomer was identified by means ofchiral GC analysis.

b) by Means of Electrodialysis (Direct Isolation of (S)-2,2-HTFMPS)

A reaction mixture containing (S)-2,2-HTFMPA and (R)-2,2-HTFMPS(obtained from Example 4.3) was subjected to ultrafiltration to removecellular material. The resulting solution was subjected toelectrodialysis. (R)-2,2-HTFMPS and all buffer salts migrated throughthe membrane. After electrodialysis had ended, a solution of pure(S)-2,2-HTFMPA (2342.2 g) was obtained. This solution was distilled at135° C. and 20 mbar, until 447 g of product were obtained. 32.7 g ofsolid NaOH (0.8 mol) were then added, and the reaction mixture wasrefluxed for 3 hours. After this time, the (S)-2,2-HTFMPA had beenconverted completely into (S)-2,2-HTFMPS. The solution was cooled to atemperature of below 25° C., and the pH was brought from 13.8 to 1.0using 93.6 g of concentrated HCl. The aqueous phase was extracted twicewith ethyl acetate (500 ml). The combined organic phases were dried withNa₂SO₄ and then filtered. The solution was concentrated on a rotaryevaporator until a viscous suspension was obtained. This suspension wastreated twice with 20 ml of toluene each time, whereupon the resultingsuspension was reconcentrated. A further 10 ml of toluene were thenadded, whereupon the mixture was refluxed. The solution was cooled toroom temperature and treated with hexane (30 ml), until the productprecipitated. The suspension was cooled to −10° C. and the product wascollected by means of ultrafiltration. Drying in vacuo (temperature <35°C.) gave 14.1 g (0.0892 mol) of pure (S)-2,2-HTFMPS (ee value 99.7%),which corresponds to a yield of 35% (calculated on the basis of half thestarting material).

EXAMPLE 5

a) Chemical Hydrolysis of (S)-2,2-HTFMPA to (S)-2,2-HTFMPS

0.47 g of sodium hydroxide (11.6 mmol) were added to 5 ml of distilledwater. 650 mg (4.14 mmol) of (S)-2,2-HTFMPA were added to this, and themixture was refluxed. After 2 hours, the reaction mixture was cooled toroom temperature and the pH was brought to 1.0 using 10% strength HCl.The mixture was subsequently extracted twice with ethyl acetate (10 ml).The combined organic phases were dried over Na₂SO₄, filtered andevaporated at not more than 40° C. Drying in a vacuum oven (45 minutesat 35° C.) gave 618 mg of (S)-2,2-HTFMPS, which corresponds to a yieldof 94.4%. Only the one isomer was identified by means of chiral GCanalysis.

b) Microbiological Hydrolysis of (S)-2,2-HTFMPA to (S)-2,2-HTFMPS

Rhodococcus equi TG 328 (DSM 6710) were grown in a mineral salt mediumas described by Gilligan et al., (ibid). The washed cells at OD₆₅₀=5.0were incubated at 37° C. with an (S)-2,2-HTFMPA solution (1% in 100 mMphosphate buffer, pH 7.7). After 16 hours, GC analysis revealed that the(S)-2,2-HTFMPA had been converted quantitatively into (S)-2,2-HTFMPS.

EXAMPLE 6

6.1 Generation of a Capsule-Negative Mutant of Klebsiella oxytoca PRS1

Klebsiella oxytoca PRS1 formed a slime capsule which conferredunfavourable characteristics on the strain during fermentation. Acapsule-negative strain was advantageous for cell separation andsubsequent work-up.

Capsule-negative mutants were isolated by means of acridine ICR 191 (J.H. Miller Experiments in Molecular Genetics, Cold Springs Harbor, 1972)as described below.

Klebsiella oxytoca PRS1 was inoculated into mineral salt mediumcontaining 0.2% of glucose in the presence of acridine ICR 191 andincubated overnight at 30° C. This culture was subsequently subculturedin fresh medium and again incubated overnight at 30° C. The culture wasdiluted and plated onto nutrient agar. Non-slimy colonies were pickedand checked. The mutants were isolated at a frequency of 0.18%. Anexample of such a mutant is Klebsiella oxytoca PRS1K17 (DSM 11623). Thismutant shows the same growth behaviour as the wild type. The(R)-specific enzyme has the same activity as in Klebsiella oxytoca PRS1,but the strain does not form a slime capsule. This mutant was used forenzyme characterization and gene cloning.

6.2 Preparation of Chromosomal DNA of Klebsiella oxytoca PRS1K17(capsule-negative mutant of PRS1)

The chromosomal DNA of a fresh overnight culture of Klebsiella oxytocaPRS1K17 (100 ml nutrient yeast broth, 30° C.) was isolated by themodified method of R. H. Chesney et al. (J. Mol. Biol., 130, 1979),161–173):

The cells which had been harvested by centrifugation (15 min, 6500×g, 4°C.) were resuspended in Tris buffer (2.25 ml, 0.05 mol/l, pH 8.0, 10%(w/v) sucrose).

After addition of 375 μl of lysozyme solution (10 mg/ml; 0.25 mol/l TrisHCl buffer, pH 8.0) and 900 μl of 0.1 mol/l EDTA, pH 8.0, the suspensionwas cooled for 10 minutes on ice. Thereupon, 450 μl of 5% (w/v) SDS and50 μl of ribonuclease (10 mg/ml H₂O) were added and the mixture wasincubated for 30 minutes at 37° C. Incubation was continued for 2 hoursafter addition of a spatula-tipful of proteinase K and 400 μl of pronase(20 ml/ml H₂O). After mixing with 4.3 g of CsCl, the mixture wascentrifuged (30 min, 40,000×g, 20° C.), treated with 250 μl of ethidiumbromide (10 mg/ml), and the mixture was centrifuged in anultracentrifuge (Vti 62.5 tubes; more than 8 hours, 246,000×g, 20° C.).The DNA band was drawn off from the tube under long-wave UV light. Afteradding 4 volumes of TE buffer (10 mmol/l Tris HCl , pH 8.0, 1 mmol/lEDTA), the ethidium bromide was extracted three times withwater-saturated n-butanol. The DNA was precipitated with isopropanol,taken up in TE buffer and incubated for 15 minutes at 65° C. Thematerial was capable of being stored at 4° C.

6.3 Restriction and Ligation of the Chromosomal DNA

5 μg of Klebsiella oxytoca PRS1K17 DNA and 4.5 μg of vector DNA(pBLUESCRIPT-KS+®) were cleaved with 20 units of restriction enzymeHindIII each in a total restriction buffer volume of 100 μl (6.5 hoursat 37° C.). The DNAs were precipitated with ethanol and dried in theSpeed Vac^(R) concentrator. The precipitates were taken up in theligation buffer (20 mmol/l Tris buffer, 10 mmol/l DTT (dithiothreitol),10 mmol/l MgCl₂, 0.6 mol/l ATP (adenosin triphosphate, pH 7.2) andcombined (ligation volume 100 μl).

After addition of 1 unit of T4 DNA ligase, the mixture was incubatedovernight at 13° C. The DNA of the ligation mixture was precipitatedwith isopropanol and taken up in 30 μl of water for transformation.

6.4 Transformation of E. coli XL1-Blue MRF′200 and Selection

Competent E. coli XL1-Blue MRF′® cells were transformed with theligation mixture by electroporation following the method described by S.Fiedler and R. Wirth (Analyt. Biochem., 170, 1988, 38–44).

To detect plasmid, selection was performed on nutrient agar withampicillin (100 μg/ml) and to detect “insert”, selection was performedwith 0.5 mmol/l IPTG (isopropyl-β-D-thiogalactoside) and X-Gal (30μg/ml, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) duringincubation at 37° C.

At a transformation frequency of 1.7×10⁸ cfu/ml (“colony-forming units”Δlive cells), virtually all clones carried a HindIII “insert”.

EXAMPLE 7

Screening of the Klebsiella oxytoca PRS1K17 Gene Library for the(R)-specific Amidohydrolase Gene

Clones carrying hybrid plasmids (HindIII “insert”) were checked fortheir ability to grow on minimal medium agar as described by H. Kulla etal. (Arch. Mikrobiol., 135, 1983, 1–7) with 0.4% (v/v) glycerol as the Csource, 0.2% (w/v) of (R,S)-2,2-HTFMPA as the sole N source andampicillin (5 μg/ml) for plasmid stabilization. Only clones whichcontained the intact amidohydrolase gene sad on the DNA “insert” in theplasmid were capable of utilizing (R,S)-HTFMPA as N source, convertingthe former into the desired (R)-acid and growing on this minimal medium.All clones which were selected in this manner contained a hybrid plasmidof vector pBLUESCRIPT-KS+® with a HindIII “insert” of approx. 2.73 kb.

This allowed identification of strain E. coli XL1-Blue MRF′® with theplasmid termed pPRS2a, from which plasmid pPRS2a was isolated andcharacterized in greater detail.

EXAMPLE 8

Localization of the Amidohydrolase Gene (sad) on the Cloned HindIIIfragment

8.1 Restriction Map of pPRS2a

A coarse restriction map of pPRS2a as regards XhoI, DraII, SmaI, PstI,SalI, BamHI was established by restriction analysis followingconventional procedures (Current Protocols Molecular Biology, John Wileyand Sons, New York, 1987, Section 2). The restriction map is shown inFIG. 1.

8.2 Formulation of Mixed DNA Oligomers Based on the AmidohydrolaseN-Terminal Peptide Sequence

The genetic code allowed the formulation, and synthesis using a DNAsynthesizer, of a mixed DNA oligomer for the Klebsiella oxytoca PRS1K17amidohydrolase N-terminal peptide sequence.

-   LON T-4-   5′ CAK CAK CTN ACN GAR GAR ATG CA 3′-   AS His His Leu Thr Glu Glu Met    AS=Amino Acid Sequence    8.3 “Southern Blot Hybridization” of Restriction Fragments of    Plasmid pPRS2a

The DNA fragments obtained from pPRS2a after different restrictions(BamHI, SmaI, DraII, HindIII, EcoRI) which had been separated by agarosegel electrophoresis (0.6%) were transferred to nitro-cellulose by theknown “Southern blot method” (Current Protocols in Molecular Biology,John Wiley and Sons, New York, 1987, Section 2.9 et seq.).

Also, the DNA oligomers were 3′-end-labelled with digoxigenin.Hybridization of the “Southern blots” followed the known procedure (inthe above-mentioned reference).

Hybridization with the nucleotide oligomer corresponding to theN-terminal protein sequence allowed a 1.44 kb SmaI/BamHI DNA fragment ora 1.52 kb DraII/BamHI DNA fragment to be identified on the hybrid plasmdpPRS2a.

8.4 Subcloning the Hydrolase Gene (sad)

The 1.52 kb DraII/BamHI DNA fragment, or the 1.91 kb PstI/BamHI DNAfragment, which encodes the (R)-specific amidohydrolase from Klebsiellaoxytoca PRS1K17 was inserted into equally digested vector DNApBLUESCRIPT-KS+®.

The vector pBLUESCRIPT-KS+® containing the 1.52 kb DraII/BamHI DNAfragment was termed hybrid plasmid pPRS7. The vector pBLUESCRIPT-KS+®which contained the 1.91 kb -PstI/BamHI DNA fragment was termed hybridplasmid pPRS4.

8.5 Sequencing the Hydrolase Gene (sad)

The 1.44 kb SmaI/BamHI fragment described further above under 8.3 wassubjected to fluorescence sequencing using Sanger's dideoxy method(modified) with the aid of a laser fluorescence DNA sequenator. In thismanner, the nucleotide sequence termed SEQ ID No. 1 was determined, fromwhich the amino acid sequence for the amidohydrolase, which is shownseparately under SEQ ID No. 2, is derived.

EXAMPLE 9

Determination of the Activity of the (R)-Amidohydrolase Clones

The determination of the activity was carried out similarly to asdescribed in Example 4.2.

The results with E. coli/pPRS1b and E. coli/pPRS 2a as examples areshown in Table 2.

Hydrolase activity (S)- (R)-amide amide Hours Clone g/l g/l (h) E. coliXL1-Blue 5.35 5.92 0 MRF′ ®/ pPRS1b (EcoRI clone) E. coli XL1-Blue 0.005.84 4 MRF′ ®/ ~Initial activity pPRS1b (EcoRI clone) (37° C.) 0.29 g/l/h/OD_(650 nm) E. coli XL1-Blue 5.66 5.92 0 MRF′ ®/ pPRS2a (HindIIIclone) E. coli XL1-Blue 0.00 6.20 8 MRF′ ®/ pPRS2a (HindIII clone)~Initial activity (37° C.) 0.13 g/l/ h/OD_(650 nm)

EXAMPLE 10

Enzyme Purification and Enzyme Characterization

10.1 Enzyme Purification

During purification, the active fractions were determined bycolorimetry. The activity of the cell-free extract and of the pureenzyme was then determined by the GC method. Klebsiella oxytoca PRS1cells (200 ml, OD₆₅₀=21 in 100 mM phosphate buffer, pH 7.5) weredisrupted by passing 3 times through a French press at 19000 psi (1309bar). Benzonase (1 μl×30 ml extract⁻¹) was added, and the extract wasthen centrifuged for 15 minutes at 100000×g. The super-natant (2.94mg×ml⁻¹) was heated for 10 minutes at 80° C., and the precipitatedprotein was then removed by centrifugation. The supernatant (170 ml,0.83 mg×ml⁻¹) was applied to a HiLoad Q-Sepharose 26/10 chromatographycolumn (Pharmacia) which had previously been equilibrated with 50 mMphosphate buffer (pH 7.5; buffer A). Unbound protein was eluted from thecolumn using 130 ml of buffer A. Then, a linear gradient (500 ml; 1 MNaCl—0 M NaCl in buffer A) was established, the flow rate being 2.5ml×min⁻¹. Fractions of 5 ml were collected and tested for activity. Themost active fractions (30–37; 40 ml) were combined, concentrated to 7.5ml by ultrafiltration, and the buffer was then exchanged for a 10 mMphosphate buffer (pH 7.5) by means of gel filtration chromatography(Sephadex G-25 M, PD 10, Pharmacia). The active fractions were thenapplied to a hydroxyapatite column (5 ml; Bio-Scale CHTI, BioRad) whichhad been equilibrated with a 10 mM phosphate buffer. Fractions of 1 mlwere collected at a flow rate of 2.0 ml×min⁻¹ using a gradient (90 ml;0.5 μM phosphate buffer 10 mM phosphate buffer, pH 7.5) and tested foractivity. Activity was shown by fractions 17–25 and 32–34. The protein(M_(r) 37000) of fraction 19 and fractions 33 and 34 was pure accordingto SDS-PAGE. The protein of fraction 20 showed a purity of over 95%.Fractions 20–25 were combined, concentrated to 200 gl and then appliedto a gel filtration chromatography column (Superose 12; Pharmacia).SDS-PAGE revealed that fractions 23–26 were pure.

10.2 Protein Sequencing

An N-terminal amino acid sequence was obtained by western blotting, andthe protein was then digested with trypsin and the peptides wereisolated by HPLC and sequenced.

N terminus: Met Lys Trp Leu Glu Glu Ser Ile Met (SEQ ID No.3) Ala LysArg Gly Val Gly Ala Ser Arg Lys Pro T3: Val Tyr Trp Ser Lys (SEQ IDNo.4) T4: Lys Pro Val Thr His His Leu Thr Glu (SEQ ID No.5) Glu Met GlnLys T5: Tyr Thr Val Gly Ala Met Leu Asn Lys (SEQ ID No.6) T6A: Met GluAsn Ala Glu Asn Ile Met Ser (SEQ ID No.7) Ile Gly Ser Ala Arg T7: TrpLeu Glu Glu Ser Ile Met Ala Lys (SEQ ID No.8) T8: Met Pro Phe Leu AsnPro Gln Asn Gly (SEQ ID No.9) Pro Ile Met Val Asn Gly Ala Glu Lys T9-2:Asp Ala Phe Glu Gly Ala Ile Asn Ser (SEQ ID No.10) Glu Gln Asp Ile ProSer Gln Leu Leu Lys T9-2: Glu Phe His Tyr Thr Ile Gly Pro Tyr (SEQ IDNo.11) Ser Thr Pro Val Leu Thr Ile Glu Pro Gly Asp Arg T11: Leu Phe IleGly Asp Ala His Ala Glu (SEQ ID No.12) Gln Gly Asp Gly Glu Ile Glu GlyThr Ala Val Glu Phe Ala T13-1: Gly Asp Val Leu Ala Val Tyr Ile Glu (SEQID No.13) Ser Met Leu Pro Arg T13-2: Gly Val Asp Pro Tyr Gly Ile Glu Ala(SEQ ID No.14) Met Ile Pro His Phe Gly Gly Leu Thr Gly Thr Asp Leu ThrAla Met Leu Asn Asp Gln Leu Gln Pro Lys10.3 Enzyme Characterization

A heat-treated cell-free extract was employed for characterizing theamidase. Cells of Klebsiella oxytoca PRS1K17 (DSM 11623) (OD₆₅₀=160)were disrupted by passing through a French press at 19000 psi (1309bar). Benzonase (1 μl×30 ml extract⁻¹) was added, and the extract wasthen centrifuged for 1 hour at 20000×g. The supernatant (approx. 20mg×ml⁻¹ protein) was heated for 10 minutes at 70° C. and theprecipitated protein was then removed by centrifugation. The supernatant(approx. 2.0 mg×ml⁻¹) was concentrated to 5.0 mg×ml⁻¹ protein and thenstored at −20° C. The heat treatment removed approx. 90% of undesiredprotein. Up to a protein concentration of 0.5 mg×ml⁻¹, the reaction ratewas in direct proportion to the protein concentration. A proteinconcentration of 0.2 mg×ml⁻¹ was therefore routinely employed in thetests. To determine the pH optimum, the concentration of(R,S)-2,2-HTFMPA (substrate) was 0.5% (32 mM) and the temperature was40° C. The buffers listed in Table 4 were employed in the test.

TABLE 4 Buffer pH 100 mM MES  6.5 100 mM HEPES  7.0; 7.5 50 mM phosphatebuffer  8.0; 8.5 50/100 mM Tris buffer  8.0; 8.5 50/100 mM borate buffer 9.0; 9.5 50/100 mM CAPS buffer 10.0; 10.5; 11.0

The effect of the temperature on the reaction was determined in 100 mMCAPS buffer (pH 10.0) at a substrate concentration of 0.5% (32 mM). Theeffect of the substrate concentration was determined at 60° C. in 100 mMCAPS buffer (pH 10.0), and the effect of methanol at 40 and 60° C. at asubstrate concentration of 1% (64 mM) in 100 mM CAPS buffer (pH 10.0).The K_(m) value of the reaction was determined using the Enzfitterprogram of Biosoft.

FIG. 4 shows the pH optimum. The pH optimum is between 9.5 and 10.5 (100mM CAPS buffer; substrate concentration 32 mM).

FIG. 5 shows the Michaelis-Menten kinetics. The K_(m) value for(R)-2,2-HTFMPA is 32 mM (60° C. in 100 mM CAPS buffer, pH 10).

FIG. 6 shows the temperature optimum. The temperature optimum is 70° C.(100 mM CAPS buffer; substrate concentration 32 mM).

FIG. 7 shows the effect of methanol. Methanol concentrations of between5 and 20% inhibit the reaction.

10.4 Enzyme Immobilization

The heat-treated cell-free extract was immobilized using Eupergit C(Röhm GmbH). To this end, Eupergit C (3.0 g) was added to 15 ml ofheat-treated cell-free extract (protein concentration: 51 mg) in 1 Mpotassium phosphate buffer (pH 8.0). The mixture was incubated for 90hours at room temperature with gentle stirring. The immobilized enzymewas filtered off and washed 4 times with 20 ml of 100 mM potassiumphosphate buffer (pH 8.0). Support-bound enzyme (49 mg) gave 9.5 g ofimmobilized enzyme (fresh weight), which was stored in 100 mM potassiumphosphate buffer (pH 10.0) at 4° C. To test the activity and stabilityof the immobilized enzyme, a small chromatography column was loaded with5 g (25 mg of protein). A peristaltic pump (0.135 ml×min⁻¹) was used tocirculate the substrate (100 ml 4% racemic amide in 100 mM CAPS buffer(pH 10)) between column and reservoir. The entire process was carriedout in a water bath. At certain intervals, samples were taken foranalysis. The enzyme was still active after 200 hours. Threebiotransformations (each with 4 g of racemic substrate, the first havingbeen carried out at 60° C. and the remaining two at 40° C.) gave a totalof 6 g of (S)-amide. At the beginning of the reaction, immobilizedenzyme (specific activity=47 μg×min⁻¹×mg protein⁻¹) was added at 60° C.,which is comparable (41%) with non-immobilized enzyme (specificactivity: 114 μg×min⁻¹×mg protein⁻¹).

1. An isolated nucleic acid molecule comprising a nucleotide sequencewhich: (i) hybridizes under stringent conditions to the nucleotidesequence of SEQ ID NO:1, wherein said stringent hybridization conditionsare hybridization at temperatures of between 60° C. and 70° C. and asalt content of 0.5 to 1.5 M; and (ii) encodes a polypeptide havingamidohydrolase activity capable of hydrolysing(R)-3,3,3-trifluoro-2-hyroxy-2-methylpropionamide of the formula:


2. An isolated nucleic acid molecule encoding the amino acid sequence ofSEQ ID NO:2.
 3. An isolated nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1.
 4. A recombinant vector comprisingthe nucleic acid molecule of claim 1, 2 or
 3. 5. The recombinant vectorof claim 4 wherein said vector is pPRS7.
 6. The recombinant vector ofclaim 4 wherein said vector is pPRS4.
 7. The recombinant vector of claim4 wherein said vector is pPRS2a.
 8. A microorganism transformed with therecombinant vector of claim
 4. 9. The microorganism of claim 8 whereinsaid microorganism is selected from the group consisting of the genusEscherichia, Pseudomonas, Comamonas, Acinetobacter,Rhizobium/Agrobacterium, Rhizobium, Bacillus, Rhodococcus orAgrobacterium.
 10. The microorganism of claim 9 wherein said Escherichiacoli is Escherichia coil DH5.
 11. The microorganism of claim 10 whereinsaid Escherichia coli is XL1-Blue MRF′®.